Fluorescence spectroscopy and imaging are being applied to a wide variety of noninvasive biomedical studies for in vitro and in vivo diagnostics. One of the challenges in tissue fluorescence is to obtain quantitative information on the molecular fluorophores in tissues despite the detected signal variability caused by tissue absorption and scattering. The overall goal of this R21 application is to develop, optimize, and validate a technology and method to rapidly, non-invasively, and quantitatively sense endogenous and exogenous fluorophores in tissues. The method combines both experimental and computational approaches, and will employ novel computational codes developed to model time-resolved excitation and fluorescent light propagation in complex tissues approximately 100 times faster than current methods. To illustrate the broad utility of the method, it will be applied to two distinct biomedical problems. A multidisciplinary team of researchers with expertise in biomedical optics, gastrointestinal endoscopy, and tissue engineering has been assembled to ensure that these goals are achieved. The specific aims are: (1) To develop a novel technology and method for rapid, quantitative, non-invasive molecular sensing in homogeneous tissues using time-resolved laser spectroscopy and the principles of tissue optics; (2) To extend the technology developed in Aim 1 to include application to inhomogeneous tissues with multiple layers, for the development and optimization of fiber-optic probes compatible with clinical endoscopy; (3) To extend the technology developed in Aim 1 to include application to tissues with multiple fluorophores per layer, to enable tissue engineers to quantitatively and noninvasively monitor the composition, structure, and function of their constructs. The technologies and methods developed and validated here are broadly applicable to a variety of applications in quantitative tissue fluorescence spectroscopy and imaging. To facilitate the generalized use of this approach, the computational codes can be efficiently disseminated to the scientific community via a download link or interactive interface on the Pl's web-site.